Differences in vimentin expression between samples were assayed using anti-vimentin rabbit monoclonal antibody (EPR3776, Abcam) and anti-rabbit IgG HRP-conjugated antibody (NA 934, Amersham Biosciences) at 1:5,000 or 1:10,000 dilution in TBS, respectively. 30 μg of whole-cell protein extracts from each treatment were separated using SDS-PAGE and transferred to nitrocellulose membranes by wet electroblotting, as described previously [14 ]. Non-specific binding sites were blocked with 5 % fat-free milk powder in TBS (pH 7.4) for 3 h. The membranes were incubated with 1:5,000 TBS-dilution of primary anti-vimentin rabbit monoclonal antibody (EPR3776, Abcam) overnight at 4 °C, then washed with TBS for 10 min three times, incubated with 1:10,000 TBS dilution of secondary anti-rabbit IgG HRP-conjugated antibody (NA 934, Amersham Biosciences) for 2 h at room temperature, and washed with TBS for 10 min three times.
Signals were visualized using the ECL chemiluminescence system (ab5801, Abcam) and detected on film (CL-XPosure Film, Thermo Scientific) using the exposition time that generated the best signal. Protein molecular weight was determined using PageRuler Plus Prestained Protein Ladder (Thermo Scientific) and assigned using Quantity One software 4.6. (BioRad). Densitometric quantifications were performed in triplicate using Quantity One software.
Signals were visualized using the ECL chemiluminescence system (ab5801, Abcam) and detected on film (CL-XPosure Film, Thermo Scientific) using the exposition time that generated the best signal. Protein molecular weight was determined using PageRuler Plus Prestained Protein Ladder (Thermo Scientific) and assigned using Quantity One software 4.6. (BioRad). Densitometric quantifications were performed in triplicate using Quantity One software.